摘要
从拟热带假丝酵母Candida pseudotropicalis 104(C104)中,通过离子交换层析和蓝色琼脂糖亲和层析分离得到依赖于辅酶NAD(H)的羰基还原酶,酶的分子量约为37.5kD。催化氧化反应的最适pH为8.5,催化还原反应的最适pH为6.0～6.5,最适反应温度均为50℃。该酶热稳定性较低,在pH7.0～8.5环境下较稳定,多数重金属离子均能导致酶活力下降。该酶的反应底物广泛,能高选择性催化还原多种氯代苯乙酮衍生物,其中还原苯乙酮、2′-氯-苯乙酮、3′-氯-苯乙酮和4′-氯-苯乙酮可产生对应的S型醇,其对映体过剩值(e.e.)均达到将近100%。底物上取代基团的位阻效应和电荷诱导效应是羰基还原酶还原底物活力大小的重要影响因素。
An NAD（H）-dependent carbonyl reductase was purified from Candida pseudotropicalis 104 （C 104） with DEAE ion exchange chromatogram and blue sepharose affinity chromatogram. The relative molecular weight of the enzyme was estimated to be 37.5 kD through SDS-PAGE. The optimal pH for enzymatic oxidation and reduction are 8.5 and 6.0-6.5, respectively, and the optimal temperature for both enzymatic reactions is 50℃. The enzyme is stable below the temperature of 30℃ and at pH between 7.0 and 8.5. Common heavy metal ions inhibit the enzyme activity. The carbonyl reductase shows to have a broad substrate specificity and high enantioselectivity. The enzyme can catalyze the reduction of acetophenone and its derivatives, such as 2＇-chloroacetophenone, 3＇-chloroacetophenone, 4＇-chloroacetophenone and 2-choloro-2＇,4＇-difluorophenethanol, to produce corresponding S- or R-alcohols. The enantiomeric excess values （e.e.） of the reduction products all can nearly reach 100%. It was found that the electronic induction and steric factor of the substituents on the substrates play an important role in the activity of the carbonyl reductase.
出处
《高校化学工程学报》
EI
CAS
CSCD
北大核心
2009年第1期92-98,共7页
Journal of Chemical Engineering of Chinese Universities
基金
国家自然科学基金(20336010,20506022)
国家973计划(2003CB716008)
工业生物技术教育部重点实验室开放课题资助项目
关键词
羰基还原酶
选择性还原
拟热带假丝酵母
分离纯化
carbonyl reductase
asymmetric reduction
Candidapseudotropicalis
purification
